This project addresses neurovascular injury during ischemic retinopathy. While this condition is associated with early neurovascular dysfunction, conventional therapies target clinically significant macula edema or neovascularization, which occur much later. Therapy to prevent/reverse ischemic retinal injury is a critical unmet need. The project goal is to delineate mechanisms of vascular and neuronal injury during retinopathy and identify novel therapeutic strategies. The investigators' studies in models of ischemic retinopathy have revealed that the urea cycle enzyme arginase is critically involved in both vascular and neuronal injury. Arginase metabolizes L-arginine to form proline, polyamines and glutamate. Excessive arginase activity reduces the L-arginine supply for nitric oxide synthase (NOS), causing it to become uncoupled and produce superoxide and less NO. Superoxide and NO react rapidly and form the toxic oxidant peroxynitrite. Glutamate and the catabolic products of polyamine oxidation can induce more oxidative stress and DNA damage, both of which can cause mitochondrial injury and premature senescence. Preliminary data show that neurovascular injury during retinopathy is associated with increased arginase expression/activity, decreased NO, polyamine oxidation, increased formation of superoxide and peroxynitrite, mitochondrial injury and premature senescence. Furthermore, the cytosolic isoform arginase 1 (A1) is implicated in premature senescence and dysfunction of vascular endothelial cells (EC), whereas the mitochondrial isoform arginase 2 (A2) appears to be involved in neuronal dysfunction/injury. Thus, it is hypothesized that activation of the arginase pathway causes neurovascular injury by uncoupling NOS and inducing polyamine oxidation and glutamate formation, thereby reducing NO and increasing oxidative stress, leading to mitochondrial dysfunction, EC senescence and vascular and neuronal dysfunction. Aim 1 will use animal and tissue culture models to test whether (A) limiting A1 expression will prevent vascular dysfunction by blocking NOS uncoupling, reducing oxidative stress and preventing mitochondrial dysfunction and senescence of ECs; (B) limiting A2 expression will prevent neuronal injury by blocking polyamine oxidation and glutamate formation, reducing oxidative stress and preventing mitochondrial and neuronal dysfunction. Aim 2 will determine the effects on neurovascular dysfunction and injury of novel therapies designed to limit arginase activity, restore NO availability and reduce oxidative stress. Innovation: This application will, for the firt time, investigate the role of arginase in retinal neurovascular injury. The studies will use molecular approaches to manipulate A1 and A2 expression in combination with real-time vascular imaging, electroretinography and morphometric analyses of neuronal and vascular injury. Therapeutic effects of limiting arginase activity and increasing NO will also be tested. Th research is expected to significantly advance the mechanistic understanding of retinal neurovascular injury and facilitate development of novel strategies for prevention and treatment of ischemic retinopathy.